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FisheriesScience 64(4), 621-626 (1998)

Activity and Expression of Glutamate Oxaloacetate during the Reproductive Cycle of a Fresh Water Fish Labeo rohita

Anand Shanker Srivastava*1, Ichiro Oohara*2,•õ , Tohru Suzuki*2, and Surendra Nath Singh*1 *1Centre of Advanced Study in Zoology , Banaras Hindu University, Varanasi-221 005, India *2Metabolism Section , Fish Nutrition Division, National Research Institute of Aquaculture, Nansei, Watarai, Mie, 516-0193, Japan

(Received February 1, 1998)

The activities of glutamate oxaloacetate transaminase (GOT) were measured spectrophotometrical ly from five tissues (, , , brain, and ovary) of a non air-breathing, herbivorous fresh water fish Labeo rohita. The activities were measured separately for cytosolic (c-) GOT and mitochon drial (m-) GOT. For all tissues examined, the maximum activities were observed in the spawning phase, followed by the pre and post-spawning phases for both c and m-GOTs. The native agarose gel elec trophoretic pattern revealed two GOT forms of different charges throughout the pre- to post-spawning phases. However, the spawning phase was marked by the expression of an additional anionic form of GOT in all tissues studied. These results indicate a distinct utilization of aspartate particularly in the spawning phase in fish via transamination reactions.

Key words: aspartate aminotransferase, GOT, isoenzymes, GPT, AspT, reproductive cycle, fish

Glutamate oxaloacetate transaminase (GOT or L-aspar rather than the kidney, gill, gut, brain, and spleen for na tate 2-oxoglutarate aminotransferase, EC: 2.6.1.1) is also tive and cultured fish, and in general, protein rich diets called aspartate aminotransferase. This catalyzes have been found to increase the GOT activity in fish .23-26) the reversible transamination reaction between L-aspartate Marquez27) measured the GOT activity in the pre-spawn and 2-oxoglutarate, and between glutamate and oxaloa ing, spawning and post-spawning phases of pink salmon cetate.The existence of GOT was first demonstrated in fish and found the highest GOT activity in the spawning phase. by Cohen.1) Electrophoretically, the enzyme exists in two In Hypophthalmichthys molitrix, Ctenopharyngodon idel major forms. A soluble or cytosolic form (c-GOT; anodal) la, and Cyprinus carpio, the highest enzyme activity was and a mitochondrial form (m-GOT; cathodal) have been obtained in the matured adult as compared to others .28) reported in most of the animals.2,3) Both the isoenzymes However, how GOT is controlled by the metabolic de participate in of the and citric mand during the reproductive stage of fish has not been fo acid cycles. Along with the both are cused on much. The aim in this paper is to clarify the also involved in the malate-aspartate shuttle.4,5) Particu changes of the activities of c-GOT and m-GOT separately, larly the cytosolic isoenzyme is predominantly involved in during the reproductive stage of the teleostean fish. Fur the process of gluconeogenesis.6l Both the isoenzymes are ther, the data of the occurrence of isoforms of GOT dur coded by the nuclear genome.7) It has been found that dis ing the reproductive stages are also given, and the relation tinct genes are responsible for coding c-GOT and m- between the activity change and the isoform expression is GOT.8-11)Multiple sub-forms of c-GOT and m-GOT were discussed. As a model species, Labeo rohita (herbivorous found in both crude extracts and purified preparations of and non air breathing) has been chosen, because this fish is chicken heart .12,13)The c-GOT has been fractionated into one of the most economically important South-East Asian three sub-forms in case of the pig enzyme .14-16) fresh water fish, which has high food values. Many works have been done on the regulation of the ac tivity of the c-GOT by hormones and in response to nutri Materials and methods tional status.17,18) It has been shown that the activity of c-GOT in liver and kidney is increased by glucocorti The females of Labeo rohita were collected from the coids.6,19)This result is consistent with the roles of these hor River Ganga in Varanasi during April-May for pre-spawn mones in gluconeogenesis .20,21)Arbuzzese et al.22) studied ing, June-July for spawning and August-September for the relationship between the activities of c-GOT and m post-spawning phases of the reproductive cycle as de GOT in various tissues of rats as a function of age . They- scribed by Medda et al.28) They were 20 to 22cm long, and suggested that mRNAs for the two isoenzymes are translat their weights were 150 to 200g. The captured individuals ed in a tissue and age related manner. The enzyme has were acclimatized in laboratory tanks for seven days, been found to exhibit the highest activity in the heart being fed on a diet made of rice, mustard oil cake, and

1 Corresponding author: Tel. +81-5996-6-1830, Fax. +81-5996-6-1962. 622 Srivastava et al. almond nut powder (2:1:1) once in a day, 2 to 5% of their buffer (100mM glycine, 2.9mM Tris, pH 7.0). Each body weights. sample containing an equal amount of proteins (30 Fish were then sacrificed at a fixed time of the day (10 to g/ml) was loaded in a lane of gel. Electrophoresis wasƒÊ 12 o'clock in the morning) for each experiment in order to carried out in a cold room at 4•Ž for 90 minutes with a avoid the effect of circadian rhythms, if any. After killing constant current of 10mA. The gel was removed and kept the fish, tissues were collected into an ice-cold 0.69% NaCl at room temperature for four to five minutes before the solution, and immediately used for assay. For each experi specific staining of GOT. ment, 10 to 12 individuals were sacrificed. The specific staining of GOT was done by the method of Rej et al.32) It was based on the coupled reaction catalyzed Chemicals by . The reaction produces a typi Analytical grade chemicals were used throughout the ex cal farmazan colour in the presence of NBT and PMS in periments. L-Aspartate, 2-oxoglutarate, the reduced form the gel. The specific staining mixture prepared in 100mm of nicotinamide adenine dinucleotide (NADH), malate de Tris/HCl buffer (pH 8.1) contained 5mM L-aspartate, 4 hydrogenase (MDH), glutamate dehydrogenase (GDH), mM 2-oxoglutarate, 160mM NAD+, and 30 U/ml of GDH. nitro blue tetrazolliium (NBT), and phenazine methosul This mixture was poured directly onto the gel in a tray. fate (PMS) were purchased from Sigma Chemical Co., The gel was incubated at 37•Ž for 12 hours. To eliminate U.S.A. Triple-distilled water was used in the preparation the background stain, the gel was washed in distilled of all reagents. water.

Preparation of tissue homogenate Statistical analysis Fish were sacrificed and tissues (liver, kidney, heart, For both the activity and the specific activity, the brain, and ovary) were removed immediately and washed average values and the standard errors were obtained from in the ice-cold 0.69% NaCl solution. Tissues were cleaned the four values of four individuals for each tissue. The sig with filter paper. Ten percent (w/v) homogenates of tis nificance of the difference of the average activities among sues were prepared in ice-cold 0.25 mt sucrose solution us the three phases were examined with the use of ANOVA. ing a Potter-Elvehjem homogenizer with a teflon pestle. The uniformity of the standard errors among the three phases were confirmed by F test. The significance of the Separation of mitochondrial and cytosolic fractions difference of the average activities by pair-wise comparison Mitochondrial and cytosolic fractions were separated among the three phases was examined by Student's t-test. following the centrifugal method by Boyde and Hui.291 Each tissue homogenate prepared was centrifuged at Results 700•~g for 15 minutes at 4•Ž in a Beckman centrifuge (model J2-21M). The supernatant obtained was cen The activity profiles during the reproductive cycle of L. ro trifuged at 14,000•~g for 30 minutes. Carefully decanted hita for several tissues supernatant containing microsomes was used for the assay Figure 1(a) and (b) show the activity profiles (U/g wet of c-GOT. weight) of c and m-GOTs, respectively, from the liver of The pellet was suspended in potassium phosphate buffer L. rohita at the three reproductive phases. It is apparent (0.01 m, pH7.4) and used for the assay of m-GOT. M that the activity was the highest at the spawning phase in GOT was then prepared as described in Boyde and Hui,29)- both c-GOT and m-GOT. It seems that the activity is a lit by low osmopressure punctuation. tle higher in the pre-spawning phase than in the post spawning phase. Similar tendencies are observed for the Assay of glutamate oxaloacetate transaminase (GOT) specific activities (U/mg protein) for both the c and m- The activities of c-GOT and m-GOT were assayed spec GOTs for the liver (Fig. 1(c)). trophotometrically by the method of Sizer and Jenkins.301 Table 1 shows the activities (unit/g wet weight) and the The reaction mixture was made by mixing 0.3ml of 1.0 M specific activities (unit/mg protein) of c-GOT in liver, potassium phosphate buffer (pH 7.4), 0.1ml of 100 units/ heart, kidney, brain, and ovary during pre-spawning, ml MDH , 0.2ml of 4 mg/ml NADH, 0.2ml of 0.25 M 2 spawning, and post-spawning phases. Liver had the oxoglutarate, 0.2ml of 0.2 M L-aspartate, and a suitably - highest activity, followed by heart, brain, kidney, and ova diluted tissue homogenate, and finally the total volume ry in the three phases. However, for the specific activity, was adjusted to 3.0ml by adding distilled water. The reac brain showed the highest activity, followed by liver, heart, tion was started by the addition of 2-oxoglutarate at 25 •Ž, ovary, and kidney in the spawning phase. In all the tissues and the decrease in the optical density (O.D.) was recorded studied, the spawning phase was marked with the highest at 340 run. One unit of enzyme activity was defined as the activity of the enzyme, followed by pre-spawning, and the amount that converts one umol of NADH per min at least activity was observed for the post-spawning phase. 25•Ž. The activity was presented per g wet weight of the tis The test of the differences of the values of the activities or sue examined and per mg of protein. the specific activities among the three phases (Student's t The concentration of protein in each tissue sample was test, Table 1) indicates that differences between the spawn- determined by the method of Lowry et al.31) ing phase and others are statistically significant for c-GOT in all the tissues. Gel electrophoretic separation of GOT Table 2 shows the profile of m-GOT in the five tissues of The separation of GOT was done by agarose gel elec the fish. Both the activity (unit/g wet weight) and the trophoresis. A 1.5% agarose gel was prepared with Tris/ snecific activity (unit/me nrotein) in all the tissues are max Phase-dependent GOT Expression in Labeo rohita 623

Fig. 1. Profiles of c-GOT and m-GOT activities for the liver of L. rohita during the three different reproductive phases. (a), c-GOT activity (U/g wet weight); (b), m-GOT activity (U/g wet weight); and (c), c-GOT (-•¡-) and m-GOT (-• -) specific activities (U/mg protein). The vertical bar shows the standard error of the average value.

Table 1. Activity and specific activity of c-GOT in different tissues during pre-spawning, spawning, and post-spawning phases of L. rohita.

, P<0.05;**, P<0.01; ***, P<0.001; N.S., not significant. Each value is presented with the form of the average •} the standard error (n=4).

i mum in the spawning phase among the three phases . The phases. Whereas, the specific activities in liver, heart, and activity is the highest in liver , followed by kidney, brain, brain at the pre-spawning phase were significantly higher heart and ovary at the spawning phase . Activity levels are than at the post-spawning phase. Differences of the activi significantly different among all the three reproductive ties and specific activities by pairwise comparison between Phases in all the tissues. Specific activity of enzyme was the spawning phase and the other two phases are statistical found to be maximum in liver , followed by brain, heart, ly significant for almost all tissues examined, as revealed ovary and kidney . In kidney and ovary, almost the same by Student's t-test (Table 2), except for the combination of specific activities were found at the pre and post-spawning the specific activities of pre-spawning and spawning phase

* 624 Srivastava et al.

Table 2. Activity and specific activity of m-GOT indifferent tissues during pre-spawning, spawning, and post-spawning phases of L. rohita.

* , P<0.05; **, P<0.01; ***, P<0.001; N.S., not significant. Each value is presented with the form of the average •} the standard error (n=4).

Fig. 2. Specific stains of GOT after native agarose gel electrophoresis of crude homogenates from (1) liver, (2) heart, (3) kidney, (4) brain, and (5) ovary of L. rohita during the three different reproductive phases. (A), pre-spawning phase; (B), spawning phase; (C), post-spawning phase.

for the liver. reproduction stage for all five tissues of L. rohita (Table 1 and 2). On the other hand, Sarkar et al.33) showed that Gel electrophoretic separation of GOT isoenzymes spawning phase is marked by less protein synthesis, Figure 2 exhibits the specific staining of GOT isoen decrease in food intake and increase in energy production zymes after separation of the crude homogenates on native for active breeding. Therefore, such a significant increase agarose gels. It is notable that GOT exists as three molecu in both c and m-GOT activities may indicate an active lar forms during spawning phase whereas, in pre and utilization of L-aspartate via transamination reaction par post-spawning phases, GOT exists as only two molecular ticularly during the spawning phase of this fish . forms. These results are also consistent with the report that the spawning phase is marked by a higher energy requirement Discussion for breeding behavior in fish.34-36)Several workers have demonstrated that the levels of gluconeogenic , It is known that both the c and m-GOTs participate in glycolytic enzymes, hormones and the RNA/DNA ratioin efficient regulation of amino acid metabolism in crease in fishes and other living organisms during their and mitochondria, respectively. Along with malate de breeding seasons.37,38)It is noteworthy that the genes hydrogenase, both are also reported to be involved in regu responsible for the synthesis of GOT is controlled by corti lation of malate-aspartate shuttle.4,5) coid hormones and the level of these hormones has also In the present study, it has been clarified that both the c- been reported to be quite high during the spawning phase GOT and m-GOT exhibited the highest activities during of fish.6,19,22,39,40)This control by hormones may be one of the spawning phase among the three phases in the the possible mechanisms for alterations in GOT activity Phase-dependent GOT Expression in Labeo rohita 625

among the three phases of the fish examined. Expression of an additional GOT particularly in the spawn Furthermore, the significantly higher activities of c and ing phase is a quite important phenomenon, because this m-GOTs during the spawning phase (Table I and 2), expression coincided with the phase specific changes of the together with the high activities of glutamate pyruvate level of GOT during the three phases. In addition, the oc transaminase (GPT) of the same fish species,41) may be currence of additional GOT during the spawning phase related to co-ordination of the two enzymes to utilize two may result from the activation of an additional gene of common co-substrates (2-oxoglutarate and glutamate) for GOT during the spawning phase, as one of the adaptive the spawning phase. It is therefore possible that the flow of mechanism. Regulation of more than two genes under metabolites through GOT and GPT is enhanced by utiliza different physiological conditions have also been reported tion of more amino acids through malate-aspartate shuttle by many workers.2,6,19,22,39,40,48,49) The study of the glucocor during the spawning phase of fish. This interpretation is ticoid dependent regulations of the activities of also supported by Sarkar et al.: they have observed that t GOT6,19,22,40) also suggests some possible roles of hormones RNA acceptance capacity for two amino acids ( on the expression of GOT as a function of reproductive and aspartate) becomes less in the spawning phase, com phases in L. rohita. It is quite likely that the reproductive pared with the pre and post-spawning phases33). This sug phase specific alteration in the activity of GOT for all tis gests that during the spawning phase the ovary utilizes sues examined is caused by regulations of both the number minimum of amino acids for the protein synthetic activity, of genes expressed and the amount of the expression of making them available for energy metabolic path via trans each gene of the GOT. amination reactions. The c-GOT activity is found to be maximum in liver fol Acknowledgments The first author is grateful to The Science and Tech lowed by heart, brain, kidney, and ovary; whereas, in case nology Agency, Japan and The University Grant Commission, India for of m-GOT, liver again shows the highest activity followed providing the funds for this work. by kidney, brain, heart, and ovary (Tables 1 and 2). The specificactivities of c-GOT and m-GOT were maximum in References brain and liver, followed by heart, ovary, and kidney dur 1) P. P. Cohen: Transamination with purified enzyme preparations. J. ing the spawning phase of fish, showing the difference in Biol. Chem., 136, 565-584 (1940). transamination status of each tissue. As explained earlier 2) E. J. Shrawder and M. M. Carrion: Simultaneous isolation and in case of GPT,41) the liver is the central tissue of all meta characterization of chicken supernatant and mitochondrial isoen bolic activities, and the heart works more actively when zyme of aspartate transaminase. J. Biol. Chem., 248, 2140-2146 more energyys required. Obviously, both the c and m (1973). GOTs seem to display well co-ordinated controls of favora- 3) A. E. Braunstein and E. E. Snell: In "" (ed. by P. ble supply of energy in all the tissues in this fish during the Christen and D. E. Metzler) Wiley and Sons, New York, 1985, pp. 2-35. spawning phase. In turn, according to Sarkar et al.,33) 4) A. J. L. Cooper and A. Meister: In "Transaminases" (ed. by P. GOT also controls direct utilization of amino acid precur Christen and D. E. Metzler) Willy and Sons, New York, 1985, pp. sors for protein synthesis during the resting and pre-spawn 534-563. ing phase of fish. Considering all these together, the levels 5) P. Christen, U. Graf-Hausner, F. Bossa, and S. Doonan: In "Trans of transaminases seem to represent metabolic status in fish aminases" (ed. by P. Christen and D. E. Metzler) Willy and Sons, tissues as a function of different life stages including the New York, 1985, pp. 173-185. three reproductive phases. 26.27.42) 6) Y. Horio, H. Fukui, M. Taketoshi, T. Tanaka, and H. Wada: In duction of cytosolic aspartate aminotransferase by glucagon in pri Although the brain exhibits lower activities of both c mary cultured rat hepatocytes. Biochem. Biophys. Res. Commun., GOT and m-GOT than liver, heart, and kidney (Tables - 1 153, 410-416 (1988). and 2), its significantly higher specific activity during the 7) S. Doonan, F. Martini, S. Angelaccio, S. Pascarella, D. Barra, and spawning phase than during the other two phases may F. Bossa: The complete amino acid sequence of cytosolic and reflect the metabolic significance of GOT in the brain tis mitochondrial aspartate aminotransferase from horse heart, and in sue of this fish at the spawning phase. It has been reported ference on evolution of the isoenzymes. J. Mot. Evol., 23, 328-335 that transaminases help in making sufficient glutamate in (1986). 8) P. J. Morin, G. S. Subramanian, and T. D. Gilmore: AAT1, a gene the brain as an important precursor of neurotransmit encoding a mitochondrial aspartate aminotransferase in Sac ter.43,441Also, it has been observed that glutamate left in the charomyces cerevisiae. Fish Physiol. Biochem., 1171, 211-214 brain may be consumed in a cycle for energy (1992). production.41,46) 9) Y. Elgersma, C. W. T. Van Roermund, R. J. A. Wanders, and H. The activities and the specific activities of c-GOT and F. 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Wanders: Cytosolic aspartate aminotransferase encod represents a peculiar migration pattern with different ed by the AA T2 gene is targeted to the peroxysomes in oleate-grown Saccharomyces cerevisiae. Eur. J. Biochem., 247, 972-980 (1997). forms of GOT in a phase dependent manner (Fig. 2). Exis 12) L. H. Bertland and N. O. Kaplan: Chicken heart soluble aspartate tence of one cathodal and one anodal form in all the tis aminotransferase. Purification and properties. Biochemistry, 7(1), sues for three phases corroborates the earlier reports. 12,1) 134-142 (1968). 626 Srivastava et al.

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